Abstract

Steroids have been proposed as endogenous ligands at σ receptors. In
the current study, we examined the ability of steroids to regulate
N-methyl-d-aspartate (NMDA)-stimulated
[3H]dopamine release from slices of rat striatal tissue. We found
that both progesterone and pregnenolone inhibit [3H]dopamine
release in a concentration-dependent manner similarly to prototypical
agonists, such as (+)-pentazocine. The inhibition seen by both progesterone
and pregnenolone exhibits IC50 values consistent with reported
Ki values for these steroids obtained in binding studies,
and was fully reversed by both the σ1 antagonist
1-(cyclopropylmethyl)-4-2′-4″flurophenyl)-2′oxoethyl)piperidine
HBr (DuP734) and the σ2 antagonist
1′-[4-[1-(4-fluorophenyl)-1-H-indol-3-yl]-1-butyl]spiro[iso-benzofuran-1(3H),
4′piperidine] (Lu28-179). Lastly, to determine whether a protein kinase
C (PKC) signaling system might be involved in the inhibition of
NMDA-stimulated [3H]dopamine release, we tested the
PKCβ-selective inhibitor
5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4 -
1][1,8]diacyclohexadecine-18,20(19H)-dione,8-[(dimethylamino)methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate
(9Cl) (LY379196) against both progesterone and pregnenolone. We found that
LY379196 at 30 nM reversed the inhibition of release by both progesterone and
pregnenolone. These findings support steroids as candidates for endogenous
ligands at σ receptors.

Since their proposal in 1976 by Martin et al.
(1976), σ receptors have
been characterized pharmacologically in bioassays and radioligand binding
studies. σ receptors bind a wide array of drugs from various classes,
including benzomorphans, guanidines, morphinans, antipsychotics, and cocaine.
However, none of these drugs is endogenous to the brain or in cells in
culture. To establish a relevance of σ receptors to physiological
function, it is important to identify an endogenous ligand.

Steroids were originally proposed as endogenous ligands at σ
receptors by Su et al. (1988).
Swartz et al. (1989)
questioned this proposal because they assumed steroids could not be produced
in the brain, and the concentration of steroids crossing the blood-brain
barrier would not suffice to occupy σ receptors. Interest in
steroid-σ receptor interactions was revived when studies showed that
steroids mediated many effects through a nongenomic mechanism
(Falkenstein et al., 2000) and
that steroids were synthesized in the brain
(Hu et al., 1987;
Jung-Testas et al., 1989;
Guarneri et al., 2000), along
with pharmacological studies on regulation of transmitter release
(Monnet et al., 1995) and
responses in hippocampal neurons (Bergeron
et al., 1996) to steroids. The steroids the Su group found that
competed best for [3H]SKF10,047 at σ1 sites were
progesterone, deoxycortisone, and testosterone. More recently, McCann and Su
(1994) examined steroid
competition at σ1 sites and found both progesterone and
testosterone had an affinity for σ1 and σ2
subtypes.

Monnet et al. (1995)
described the modulation of NMDA-stimulated [3H]norepinephrine
release by progesterone, dehydroepiandrosterone sulfate (DHEA S), and
pregnenolone sulfate (PREG S) in rat hippocampal slices. The effects were
blocked by σ receptor antagonists and progesterone acted as an
antagonist to DHEA S and PREG S. Most recently, Meyer et al.
(2002) showed that PREG S
enhances glutamate release from hippocampal slices via a
σ1-like receptor. In studies reviewed by Bastianetto et al.
(1999) and Maurice et al.
(1999), PREG S, DHEA S, and
allotetrahydroxycorticosterone show antistress, anxiolytic, and antiamnesiac
activity that is blocked by σ antagonists, as well as antisense
oligonucleotides to σ1 receptors
(Maurice et al., 2001). Taking
all these studies in consideration, steroids at this time are likely
candidates for endogenous σ receptor ligands.

Gonzalez-Alvear and Werling
(1994) first demonstrated
regulation of NMDA-stimulated [3H]dopamine release from rat
striatum by σ receptor ligands, including (+)-pentazocine,
(+)-SKF10,047, and BD737. The inhibition produced by low concentrations of
these ligands was reversed by the σ1 antagonist DuP734
(Gonzalez-Alvear and Werling,
1994,
1995). A second phase of
inhibition produced by higher concentrations of (+)-pentazocine was reversed
by nonsubtype-selective σ receptor antagonists, indicating the
participation of σ2 receptors. Studies by Izenwasser et al.
(1998) revealed that
amphetamine-stimulated [3H]dopamine release can be modulated by
σ2 receptor agonists and antagonists in vitro. In our current
study, we examined the ability of steroids to regulate NMDA-stimulated
[3H]dopamine release from rat striatal slices via σ
receptors. If steroids are indeed the endogenous ligand for σ receptors,
they should affect neurotransmitter release and signaling similarly to
prototypical σ ligands. We have previously demonstrated that
σ1 agonist-mediated inhibition of NMDA-stimulated
[3H]dopamine release is mediated by a PKC signaling system, likely
involving the β isoform. σ receptor regulation of release is
abolished by pretreatment with phorbol 12-myristate 13-acetate, as well by
treatment with a PKCβ or a PLC inhibitor
(Nuwayhid and Werling, 2003).
Therefore, we tested whether the same PKC pathway is involved in regulation of
stimulated dopamine release by steroids via σ receptors by using a
PKCβ inhibitor.

Measurement of Stimulated [3H]Dopamine Release from Striatal
Slices. All experiments were carried out in accordance with the guidelines
and the approval of the George Washington University Institutional Animal Use
and Care Committee. Male Sprague-Dawley rats (Hilltop Lab Animals, Scottsdale,
PA), weighing 200 to 225 g, were sacrificed by decapitation, and brains
removed to ice. Striata were dissected, chopped in two planes at right angles
into 250 × 250 μm strips with a Sorvall T-2 tissue sectioner, and
suspended in modified Krebs-HEPES buffer (MKB; 127 mM NaCl, 5 mM KCl, 1.3 mM
NaH2PO4, 2.5 mM CaCl2, 15 mM HEPES, 10 mM
glucose, pH adjusted to 7.4 with NaOH) by titration through a plastic pipette.
Buffers were oxygenated throughout the experiments and brain slices were kept
at a constant temperature of 37°C. After three washes in MKB, tissue was
resuspended in 20 ml of MKB and incubated for 30 min with 0.1 mM ascorbic acid
and 15 nM [3H]dopamine. Tissue was then washed twice with 20 ml of
MKB and once in 20 ml of MKB containing 10 μM nomifensine and 1 μM
domperidone. These drugs were included in all subsequent steps to prevent
reuptake of and feedback inhibition by the released [3H]dopamine.
Tissue was suspended a final time in 7.5 ml of MKB, containing 10 μM
nomifensine and 1 μM domperidone, and distributed in 275-μl aliquots
between glass fiber discs into chambers of a superfusion apparatus (Brandel,
Inc., Gaithersburg, MD). MKB was superfused over tissue at a rate of 0.6
ml/min. A low stable baseline release of approximately 1.3%/2 min collection
interval was established over a 30-min period. Tissue was then stimulated by a
2-min exposure to 25 μM NMDA (S1). The mean fractional release (percentage)
produced in the S1 stimulus interval was 11.9 ± 1.2%. Inflow was then
returned to nonstimulating buffer during a 10-min interstimulus interval
(ISI). If a steroid, σ antagonist, cholesterol, mifepristone, or
trilostane was being tested, it was included at this time. The inclusion of
σ antagonist drug in the buffer did not significantly affect basal
release (per 2-min collection interval: no antagonist 1.3 ± 0.33,
n = 3; 100 nM DuP734, 1.2 ± 0.24, n = 3; 1 nM
Lu28-179 1.4 ± 0.14, n = 3). Neither did inclusion of steroid
affect basal release significantly (progesterone, 1.3 ± 0.24,
n = 4; pregnenolone, 1.6 ± 0.09, n = 3). Tissue was
then exposed to a second stimulus (S2) identical to the first except in the
presence of a steroid, trilostane, mifepristone, or cholesterol, as
appropriate. In the experiments testing the PKC inhibitor LY379196, the drug
was present throughout S1, ISI, and S2. Inflow was once again returned to
nonstimulating buffer before extraction of the remaining radioactivity in the
tissue by a 45-min exposure to 0.2 N HCl at a reduced flow rate. Superfusates
were collected at 2-min intervals in scintillation vials with the glass fiber
filter discs and tissue collected into the final vials. Released radioactivity
was determined by liquid scintillation spectroscopy.

All data were statistically analyzed as ratios (S2/S1) before conversion to
percentage of control values for presentation. The ratio of S2/S1 in the
absence of any test drug was 0.54 ± 0.07 (n = 10). An
enhancement by test drug would result in a higher ratio and an inhibition in a
lower ratio. In this way, differences in response between tissue samples are
taken into account and therefore, do not affect the comparison of treatments.
In the results, data are expressed as radioactivity released above baseline
during the collection interval as a fraction of the total radioactivity in the
tissue at the beginning of the collection interval (fractional release,
percentage) or as a percentage of the radioactivity released by the control
stimulus (percentage of control-stimulated release). Data are presented as a
percentage of control-stimulated release for facilitation of comparison across
experiments. Under the experimental conditions used, the released
radioactivity has been shown to be primarily dopamine
(Werling et al., 1988). All
statistical analyses were performed by one-way factorial analysis of variance
with post hoc Dunnett's. Statistical significance is considered at p
< 0.05.

Results

To examine the possibility that steroids could act similarly to identified
agonists or antagonists at σ receptors, we tested several steroids for
their ability to regulate NMDA-stimulated [3H]dopamine release via
σ receptors from rat striatal tissue. We first tested single
concentrations of progesterone, pregnenolone, DHEA, and testosterone to
determine whether they had any effect on NMDA-stimulated
[3H]dopamine release. As seen in
Fig. 1, neither DHEA at 10
μM (Ki at σ receptors = 3.7 μM,
undifferentiated for subtype; Klein and
Musacchio, 1994) nor testosterone at 3 or 10 μM
(Ki = 1 μM, undifferentiated for subtype;
Su et al., 1988) showed any
significant difference from control-stimulated release.

Lack of effect of 10 μM DHEA, 3 or 10 μM testosterone on
NMDA-stimulated [3H]dopamine release. Release of
[3H]dopamine from slices of rat striatal tissue was stimulated by
25 μM NMDA or 25 μM NMDA in the presence of DHEA or testosterone as
indicated. Statistical analysis by analysis of variance, performed on
untransformed data (ratios of S2/S1) indicated no significant difference in 25
μM NMDA alone compared with 25 μM NMDA in the presence of DHEA or
testosterone. Data are expressed as percentage of control NMDA-stimulated
release. n = 3 independent experiments in which each treatment was
tested in triplicate. Note break in y-axis.

In preliminary experiments, progesterone at 3 μM and pregnenolone at 1
μM showed an inhibition of NMDA-stimulated [3H]dopamine release.
We then constructed concentration-response curves for both these steroids. As
seen in Fig. 2A, progesterone
inhibited NMDA-stimulated [3H]dopamine release in a
concentration-dependent matter. Progesterone has a Ki
value of 270 nM at σ receptors, undifferentiated for subtype
(Su et al., 1988). The
IC50 for inhibition of release was similar to this value, lying
between 100 and 300 nM. Progesterone significantly inhibited release at 300 nM
and 1 μM. At 1 μM, the highest concentration tested, progesterone
inhibited release approximately 30%, similar to the maximum inhibition
produced by (+)-pentazocine that was attributable to σ receptors
(Gonzalez-Alvear and Werling,
1994). To confirm whether the inhibition by progesterone was
mediated through σ receptors, we tested the σ1
receptor-selective antagonist DuP734 (100 nM) and the σ2
receptor-selective antagonist Lu28-179 (1 nM) against 1 μM progesterone
(Fig. 2A). Both DuP734 and
Lu2-1798 completely reversed the inhibition of release by progesterone to
values slightly above control-stimulated release. The elevation above control
is not significant for Lu28-179, and although the value for DuP734 achieves
statistical significance as differing for NMDA alone, is associated with a
high error value.

Concentration-response curves for progesterone (A) and pregnenolone (B) on
NMDA-stimulated [3H]dopamine release, and reversal by σ
receptor antagonists. Release of [3H]dopamine from slices of rat
striatal tissue was stimulated by 25 μM NMDA alone or 25 μM NMDA in the
presence of progesterone or pregnenolone as indicated, with or without the
σ1 antagonist DuP734 (100 nM) or the σ2
antagonist Lu28-179 (1 nM). Data are expressed as percentage of control
NMDA-stimulated release. *, significantly different from NMDA alone
at p < 0.05. #, significantly different from NMDA in the presence
of progesterone or pregnenolone as indicated, p < 0.05. n
≥ 3 independent experiments in which each treatment was tested in
triplicate. Note breaks in y-axes.

Next, we constructed a concentration-response curve for pregnenolone. As
seen in Fig. 2B, the
IC50 value for inhibition of release was between 300 nM and 1
μM. Pregnenolone at 3 μM inhibited release approximately 25%. As with
progesterone, we also tested pregnenolone (3 μM) with both the
σ1 antagonist DuP734 (100 nM) and the σ2
antagonist Lu28-179 (1 nM). Both DuP734 and Lu28-179 completely reversed the
inhibition by pregnenolone (Fig.
2B) to levels not different from control-stimulated release.

Because DHEA and testosterone have affinity for σ receptors, but did
not show any inhibition of NMDA-stimulated [3H]dopamine release, we
tested whether they might behave as σ antagonists in our assay. We
tested DHEA and testosterone in combination with both progesterone and
pregnenolone to examine whether they reversed the inhibition of
NMDA-stimulated [3H]dopamine release seen by both progesterone and
pregnenolone. Neither DHEA (10 μM) nor testosterone (3 μM) had any
effect on the inhibition of [3H]dopamine release seen by either
progesterone (Fig. 3A) or
pregnenolone (Fig. 3B).

Lack of effect of testosterone or DHEA on the inhibition of NMDA-stimulated
[3H]dopamine release by 1 μM progesterone (A) and 3 μM
pregnenolone (B). Release of [3H]dopamine from slices of rat
striatal tissue was stimulated by 25 μM NMDA alone or 25 μM NMDA in the
presence of agonist steroid, as indicated, with or without potential
antagonist steroid testosterone (3 μM) or DHEA (10 μM). Data are
expressed as percentage of control NMDA-stimulated release. *,
significantly different from NMDA alone at p < 0.05. Neither the
inclusion of testosterone nor DHEA significantly changed the inhibition
produced by progesterone or pregnenolone alone. n = 3 independent
experiments in which each treatment was tested in triplicate. Note breaks in
y-axes.

To determine whether PKCβ is involved in the inhibition of
NMDA-stimulated [3H]dopamine release, we constructed dose-response
curves for the PKCβ-selective inhibitor LY379196 against both
progesterone and pregnenolone. We have previously shown that LY379196 blocked
(+)-pentazocine-mediated inhibition of NMDA-stimulated [3H]dopamine
release in a concentration-dependent manner
(Nuwayhid and Werling, 2003).
LY379196 has Ki values at βI and βII isozymes of
18 and 16 nM, and all other isozymes, including PKCα, PKCδ,
PKCγ, PKCϵ, and PKCη >300 nM (Louis Vignati, personal
communication). As seen in Fig.
4A, LY379196 completely reversed the inhibition of release by 1
μM progesterone at both 30 and 100 nM. At 30 nM, LY379196 completely
reversed the inhibition of release by 3 μM pregnenolone
(Fig. 4B).

Reversal of steroid-mediated inhibition of NMDA-stimulated
[3H]dopamine release by the PKCβ-selective inhibitor LY379196.
Release of [3H]dopamine from rat striatal slices was stimulated by
25 μM NMDA alone or 25 μM NMDA in the presence of 1 μM progesterone
(A) or 3 μM pregnenolone (B), as indicated, with or without the indicated
concentration of LY379196. Data are expressed as percentage of control
NMDA-stimulated release. *, significantly different from NMDA alone
at p < 0.05, n = 3 independent experiments. #,
significantly different from 1 μM progesterone or 3 μM pregnenolone
without PKC inhibitor at p < 0.05, n = 3 independent
experiments in which each treatment was tested in triplicate. Note breaks in
y-axes.

Progesterone and pregnenolone are synthesized from cholesterol, as
demonstrated in neural cells in culture
(Guarneri et al., 2000). We
therefore tested cholesterol to determine whether it, as the parent compound,
displayed any effects on NMDA-stimulated [3H]dopamine release.
Cholesterol (25 μM) did not affect NMDA-stimulated release of
[3H]dopamine (Fig.
5).

Lack of effect of cholesterol on NMDA-stimulated [3H]dopamine
release. Release of [3H]dopamine from slices of rat striatal tissue
was stimulated by 25 μM NMDA alone or 25 μM NMDA in the presence 25
μM cholesterol, as indicated. There was no significant difference in
release of [3H]dopamine stimulated by 25 μM NMDA alone compared
with 25 μM NMDA in the presence of 25 μM cholesterol. Data are expressed
as percentage of control NMDA-stimulated release. n = 3 independent
experiments in which each treatment was tested in triplicate. Note break in
y-axis.

Last, to verify the effects seen by pregnenolone on NMDA-stimulated
[3H]dopamine release were attributed to pregnenolone and not
dependent upon its conversion to progesterone or other metabolite, we tested
pregnenolone in the presence of 20 μM trilostane. Trilostane is an
inhibitor of 3β-hydroxysteriod dehydrogenase, an enzyme that converts
pregnenolone to progesterone (Ki = 50 nM;
Takahashi et al., 1990). As
seen in Fig. 6, inhibition of
[3H]dopamine release by 3 μM pregnenolone in the presence of
trilostane was not significantly different compared with the inhibition of
[3H]dopamine release by 3 μM pregnenolone alone. We also
verified that progesterone was acting through σ receptors and not
progesterone receptors by testing 1 μM progesterone in the presence of 10
μM mifepristone, a progesterone receptor antagonist. As seen in
Fig. 7, there was no difference
in the inhibition seen by 1 μM progesterone alone compared with 1 μM
progesterone in the presence of 10 μM mifepristone.

Lack of significant effect of trilostane on pregnenolone-mediated
inhibition of NMDA-stimulated [3H]dopamine. Release of
[3H]dopamine from slices of rat striatal tissue was stimulated by
25 μM NMDA alone or 25 μM NMDA in the presence 3 μM pregnenolone, as
indicated, with or without 25 μM trilostane. Data are expressed as
percentage of control NMDA-stimulated release. *, significantly
different from NMDA alone at p < 0.05, n = 3 independent
experiments in which each treatment was tested in triplicate. Note break in
y-axis.

Lack of effect of mifepristone on the inhibition of NMDA-stimulated
[3H]dopamine release by 1 μM progesterone. Release of
[3H]dopamine from slices of rat striatal tissue was stimulated by
25 μM NMDA alone or 25 μM NMDA in the presence 1 μM pregnenolone, as
indicated, with or without mifepristone. Data are expressed as percentage of
control NMDA-stimulated release. *, significantly different from
NMDA alone at p < 0.05, n = 3 independent experiments in
which each treatment was tested in triplicate. Note break in
y-axis.

Discussion

The identity of the endogenous ligand for σ receptors has been
equivocal. Su et al. (1988)
found that progesterone, deoxycortisone, and testosterone competed for
[3H]SKF10,047 at σ1 sites in guinea pig brain
tissue and proposed steroids as the endogenous ligands. Several studies have
now demonstrated that steroids are synthesized in the brain
(Hu et al., 1987;
Jung-Testas et al., 1989;
Guarneri et al., 2000).

Steroids exhibit genomic and nongenomic effects
(Ruppert and Holsboer, 1999).
In the current study, we examined presumably nongenomic effects of steroids on
NMDA-stimulated [3H]dopamine release in rat striatal tissue via
σ receptors. The effects on regulation of dopamine release occur within
a relatively short time frame because the length of exposure of tissue to
steroid is 12 min, not likely sufficient to produce changes in protein
expression. If steroids are endogenous ligands for σ receptors they
should behave similarly to prototypic ligands, such as (+)-pentazocine.

We found that both progesterone and pregnenolone inhibited NMDA-stimulated
[3H]dopamine release. Both inhibited release in a
concentration-dependent manner, with a maximum of about 25 to 30%, similar to
the inhibition seen by (+)-pentazocine in studies by Gonzalez-Alvear and
Werling (1994,
1995). The IC50
value of progesterone (300 nM) for inhibition of NMDA-stimulated
[3H]dopamine release was similar to its Ki
value of 270 nM in competing for binding to σ receptors
(Su et al., 1988).
Pregnenolone showed an IC50 value in the range of 300 nM to 1
μM. A Ki value at σ receptors in brain tissue has
not been reported, but we found that pregnenolone competed for
σ1 binding with a Ki value of 980
± 340 nM in SH-SY5Y cells (Werling,
2002). Su et al.
(1988) reported a
Ki value of 3.2 μM for pregnenolone sulfate binding to
σ1 receptors. These findings support that pregnenolone acts
via σ receptors to inhibit NMDA-stimulated [3H]dopamine
release.

The hypothesis that progesterone and pregnenolone act as σ agonists
in our assay was confirmed by the action of σ antagonists. The
inhibition of NMDA-stimulated [3H]dopamine release by 1 μM
progesterone and 3 μM pregnenolone was fully reversed by the
σ1 antagonist DuP734 (Ki = 10 nM)
(Culp et al., 1992) at 100 nM
and the σ2 antagonist Lu28-179 (Ki = 0.12
nM) (Moltzen et al., 1995) at
1 nM. The reversal seen by 100 nM DuP734 was somewhat above control, although
associated with a relatively high error determination. It is possible that
there is antagonism of tone exerted by endogenous steroids. However, the
antagonists had no effect on basal release, which argues against this
possibility. The action seen by both DuP734 and Lu28-179 suggest that the
inhibition of [3H]dopamine release is mediated through both
σ1 and σ2 receptor subtypes. This is a
contrast to our findings (Gonzalez-Alvear and Werling,
1994,
1995) in which components of
(+)-pentazocine-mediated inhibition were clearly attributable to either
σ1 or σ2 receptors based on reversal by
subtype-selective antagonists. However, in our assays examining the effects of
σ2 agonists on amphetamine-stimulated [3H]dopamine
release from rat striatal slices, we found similar antagonism of effect by
σ1 and σ2 antagonists
(Liu et al., 2001) to that
seen for the current steroid experiments. The σ1 and
σ2 receptors are relatively small (σ1 = 28
kDa, Hanner et al., 1996;
σ2 = 22 kDa, Hellewell
and Bowen, 1990). Perhaps upon activation of σ receptor
subtypes by progesterone and pregnenolone the receptors interact with each
other, which in turn inhibits [3H]dopamine release. In this
situation, competing off the steroid agonist with either σ1
or σ2 antagonists would prevent regulation of dopamine
release. Reports in which σ receptor protein was purified and detected
via photoaffinity labeling (Schuster et
al., 1995) or with antibody selective for σ receptor
(Hanner et al., 1996), show a
band with a molecular weight of approximately 60 kDa, which could be a dimer
of σ1 and/or σ2 receptors. Neuroactive
steroids are known to modulate the actions of GABAA and NMDA
receptors (Ruppert and Holsboer,
1999). Although it cannot be absolutely excluded that the effects
of the steroids observed in the current study involve actions via one of these
receptors, the regulation of dopamine release by the steroids is presumably
via actions at the dopaminergic nerve terminal, because σ receptors
regulating dopamine release have been localized to that location
(Gonzalez-Alvear and Werling,
1995). Regardless of whether GABAA or NMDA receptors
contribute in some way to the overall response, the effects of progesterone
and pregnenolone are both completely reversed in the current study by σ
receptor antagonists.

The concentrations of DHEA and testosterone tested were chosen based on
their reported Ki values from binding studies. Although
both competed for σ receptor binding
(Su et al., 1988;
Klein and Musacchio, 1994),
our data do not indicate that the σ receptor subtype(s) involved in the
regulation of dopamine release are sensitive to these steroids. Neither showed
an inhibition of stimulated dopamine release, and when tested for potential
σ antagonist activity, neither reversed the inhibition of
[3H]dopamine release by progesterone or pregnenolone. DHEA and
testosterone may bind to σ receptors but not have either agonist or
antagonist properties in our system. It is possible that higher or lower
concentrations could have produced effects, although this would not be
predicted based on reported affinities at σ receptors. Further
confirming that σ agonist properties are conferred only upon specific
steroids, and not the heterocyclic steroidal structure in general, was the
finding that cholesterol, the parent compound from which steroids are derived,
had no effect on NMDA-stimulated [3H]dopamine release.

We confirmed that the effects displayed by pregnenolone were due to
pregnenolone itself and not its metabolite progesterone, using trilostane, an
inhibitor of 3β-hydroxysteroid dehydrogenase enzyme that converts
pregnenolone into progesterone. Therefore, both progesterone and pregnenolone
seem to act as σ receptors agonists in the regulation of dopamine
release.

Monnet et al. (1995) showed
that DHEA S potentiated [3H]norepinephrine release from rat
hippocampal slices and this response was reversed by both σ antagonists
haloperidol and BD1063. In a later article (Monnet et al., 1996), they also
showed regulation of norepinephrine release by other steroids, but with a
different pharmacology than ours. Our data show regulation of NMDA-stimulated
[3H]dopamine release by steroids is inhibitory. This could be due
to the difference in the neurotransmitter studied or other experimental
variables. We have previously shown that σ receptors that regulate
dopamine release from striatum are located on dopaminergic nerve terminals,
but those regulating norepinephrine release are not. Therefore, the strength
of the stimulus and the circuitry involved in hippocampus is likely to be much
more complex. Monnet et al.
(1995) used a concentration of
NMDA 4 times that used by us in the current experiment and in our previous
experiments on norepinephrine. The higher NMDA concentration could activate
multiple other neurons upon which sigma receptors are located. Monnet et al.
(1995) identified that the
effects observed for some σ agonists were mediated by different
populations of σ receptor subtypes depending upon the agonist used. It
seems that our responses are due to activation of only σ1 and
σ2 subtypes only as identified by selective antagonists.

Other groups have also reported effects of steroids that were mediated by
σ receptors. PREG S increase spontaneous glutamate release via
activation of a presynaptic Gi/o-coupled σ receptor
(Meyer et al., 2002). Because
steroid sulfotransferases and sulfatases are present in the central nervous
system (Rajkowski et al.,
1997), both sulfated and nonsulfated forms could be present in the
brain and modulate neurotransmitter release. However, the permeability of
steroids is reduced by the addition of sulfate; if σ receptors are
intracellular, as suggested by McCann and Su
(1994) and Vilner and Bowen
(2000), the actions of
sulfated steroids would be limited. Unsulfated steroids would have to enter
the cell and be subsequently sulfated to become active. It is also possible
that sulfated steroids could be concentrated and stored in vesicles
(Gibbs and Farb, 2000).
However, the weak activities of sulfatases and sulfotransferases make the
conversion of steroids in vivo doubtful
(Baulieu and Robel, 1996).

Studies have now implicated the PKC signaling system in σ receptor
mediated processes. GF109203x, a PKC inhibitor, abolished σ2
receptor-mediated regulation of dopamine transporter activity
(Derbez et al., 2002).
Morin-Surin et al. (1999)
showed a parallel translocation of σ receptors and PKCβI and
PKCβII from the cytosol to the plasma membrane upon σ agonist
application. They also showed a time-dependent desensitization in the ability
of (+)-pentazocine to reduce the firing rate in rat hypoglossal neurons. This
desensitization may have been attributed to the desensitization of PKC itself.
In our studies, LY379196, a PKCβ-selective inhibitor, blocked the
inhibition of [3H]dopamine release produced by (+)-pentazocine
(Nuwayhid and Werling, 2003).
U73122, a PLC inhibitor, also blocked (+)-pentazocine-mediated regulation.
This suggests that in order for (+)-pentazocine to exert its inhibition of
dopamine release the PLC/PKC system must be intact. PKC activation has also
been linked to σ inhibition of NMDA-stimulated
Ca2+ changes in cerebellar granular cells
(Snell et al., 1994).

In the current study, we determined that the inhibition of NMDA-stimulated
[3H]dopamine release by steroids is dependent upon PKC activation.
Steroids are known to interact with the PKC pathway. Progesterone can rapidly
stimulate phosphatidylinositol bisphosphate hydrolysis, leading to the
formation of diacylglycerol and inositol triphosphate, presumably due to the
action of Ca2+-dependent PKC
(Thomas and Meizel, 1989).
Estrogen increased inositol triphosphate concentrations and stimulated
PKCα levels in membrane fraction of HEPG2 cells through a nonclassical
steroid mechanism (Marino et al.,
1998). Last, in studies by Condliffe et al.
(2001), 17-β-estradiol
regulated Cl- secretion in rat colonic epithelium, and regulation
is blocked by the chelation of Ca2+ with BAPTA or the
PKC inhibitor chelerythrine. In our study, steroids exhibited the same effects
of (+)-pentazocine in the presence of LY379196, an inhibitor of PKCβ
isozymes. This reinforces the possibility that steroids have nongenomic
actions and can signal through PLC/PKC via their actions at σ
receptors.

In conclusion, both progesterone and pregnenolone have been identified as
σ receptor agonists in the modulation of NMDA-stimulated
[3H]dopamine release. We found them to behave similarly to
prototypic ligands, such as (+)-pentazocine. In addition, we have shown that
the inhibition of NMDA-stimulated [3H]dopamine release mediated by
progesterone and pregnenolone involves a PKC signaling system most likely to
involve the PKCβ isozyme. Our findings in this study further support
steroids as candidates for endogenous ligands at σ receptors.

Acknowledgments

We thank Dr. John Puddefoot (Queen Mary and Westfield College) for the gift
of trilostane, Dr. Rob Zaczek (DuPont Merck) for the gift of DuP734, and Dr.
Connie Sanchez (H. Lundbeck) for the gift of Lu28-179. We also thank Dr. Nancy
Pilotte (National Institute on Drug Abuse) for helpful suggestions regarding
experimental design using steroids.

Footnotes

This work was supported by a grant from National Institute on Drug Abuse
(DA06667) and a Faculty Enhancement Research Award from George Washington
University Medical Center (to L.L.W.).